The present invention relates to methods for determining the reproductive quality of mammalian sperm. In particular, the present invention relates to determining reproductive quality by visualization of sperm characteristics related to the competence of sperm to initiate microtubule formation after fertilization.
Currently, there are a number of methods to assay or test for male fertility. Current tests test for sperm number, motility, and morphology, for the acrosome reaction, for the release of acrosin, and for the ability to penetrate into zona-free hamster oocytes, as well as the hemizona assay and assays for semen volume and anti-sperm antibodies. While many of these tests provide useful information, there are other mechanisms of sperm fertility failure not diagnosed by these tests, and no single test is a consistently accurate and reliable test of general sperm fertility. One of the more widely used tests, the penetration of sperm into zona-free hamster oocytes, has proven to be unreliable, providing significant percentages of both false positives (i.e. infertile men who pass the test) and false negatives (i.e. fertile men who fail the test). In addition, no present tests provide an indication of relative potency so as to indicate which sperm, e.g. from among potential donors, has greater fertilizing potential than other sperm.
Semen quality has been analyzed in a number of methods. The first parameter measured is simply volume of the ejaculate (Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction Manual, World Health Organization, 1987). The semen is also screened for anti-sperm antibodies which interfere with fertilization. Anti-sperm antibodies are evaluated in several fashions such as by sperm agglutination, sperm immobilization and immunobead binding. The last example includes some commercially available tests such as SpermCheck (Bio-Rad, Hercules Calif.) and SpermMar (Ortho Diagnostic Systems; Beerse Belgium) and may be used for direct assay (binding to sperm) or for indirect assay (presence of sperm antibodies in the serum) (Khoo, et al., Am. J. Reprod. Immunol. 26:11-16, 1991).
Semen has also been screened for the presence of tumor associated trypsin inhibitor (TATI) (Barnti, et al., Scand. J. Clin. Invest. 51(Suppl. 207):51-53, 1991). Acrosin, an enzyme important during fertilization, is a serine protease with trypsin specificity. TATI has been shown to be present in semen and shown to be inhibitor of acrosin (Hoppe-Seylers, Z. Physiol. Chem. 365:819-825, 1984).
Sperm are also directly analyzed for a number of criteria. First, the sperm may be examined for normal morphology, total number of sperm, and number of viable sperm using a light microscope (Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction Manual, World Health Organization, 1987).
Secondly, sperm may be examined for motility. Motility has been divided into two types: percentage of motile sperm vs. immotile sperm and percentage of motile sperm with forward progression vs. immotile sperm.
Two other tests of sperm activity are used, the sperm motility index, which is a measurement of disturbances in optical density of the semen (Bartoov, et al. Fertil. Steril. 56:108-112, 1991) and the resazurin reduction test, which measures reduction of the dye resazurin by a color change reaction (Glass, et al. Fertil. Steril. 56:743-746, 1991).
The integrity of sperm plasma membranes are evaluated using the hypo-osmotic swelling test (HOS-test In this test, sperm are evaluated for percentage showing swelling in hypo-osmotic media (Smith, et al., Int. J. Androl. 15:5-13, 1992). There is debate as to whether there is also significance in differential tail swelling patterns.
A popular test conducted to evaluate sperm quality is the hamster oocyte penetration test (HOP-test), sometimes referred to as the xe2x80x9chumsterxe2x80x9d assay, first referenced above. Sperm are added to media containing zona free hamster oocytes. The hamster oocytes are then evaluated for penetration by the sperm and for decondensation of the sperm nucleus. This assay also analyzes the sperm""s ability to capacitate and undergo the acrosome reaction, because these events are necessary for penetration (Yanagamachi, et al., Biol. Reprod. 15:471-476, 1976). While this test is sanctioned by the National Institutes of Health (NIH), and it is used commercially by the medical community, its predictive value is subject to question.
There are a number of industries which also evaluate sperm quality, for example, for the artificial insemination of domestic farm animals and other animals such as dogs, thoroughbreds, and llamas. In the case of domestic farm animals, the primary methods of evaluation are non-return rate, which is the percentage of bred animals (artificial insemination or natural breeding) which do not return into estrus, and the in vitro fertilization rate of homologous oocytes. In those animals which are naturally bred rather than artificially inseminated, the only evaluation is non-return rate. Therefore, there is a need in the art of reproductive biology for an assay that will evaluate the reproductive quality of a sperm sample.
There is also a need in the art for an assay that will evaluate the reproductive ability of sperm samples and identify and test for other modes of sperm reproductive failure.
The present invention describes several methods for assaying for sperm fertility and quality, all of which are based on tests for sperm capability to initiate microstructure formation in a fertilized oocyte or egg extract. The ability to test for microtubule formation initiation by sperm both allows for identification of a newly recognized form of sperm failure as well as an assay for relative sperm fertility.
Thus one aspect of the present invention is a method for evaluating relative sperm fertility. The first step of the method is to obtain a quantity of sample mammalian sperm and a sample of mature mammalian oocytes. The oocytes are fertilized with the sperm, and the cumulus cells are removed from the fertilized oocytes. The oocytes are then labelled with antibodies directed to microtubules, and the labelled microtubules are observed microscopically. The microtubule pattern is evaluated for microtubule aster size and organization emanating from the base of the sperm head.
In one preferred form of this aspect of the invention the mammalian sperm and the mammalian oocytes are of the same species. In another preferred form of the present invention the mammalian sperm and oocytes are of different species.
In another preferred form of the invention, the fertilized oocytes are labelled with anti-tubulin antibodies or antibodies to microtubule organizing antigens.
Another aspect of the present invention is a cell-free assay for sperm centrosome competence. This assay permits the convenient diagnosis of a form of sperm reproductive failure.
It is an object of the present invention to evaluate sperm samples for reproductive quality.
It is another object of the present invention to evaluate mammalian sperm to assay for causes of reproductive failure.
It is an advantage of the present invention that the reproductive quality of the sperm may be evaluated at the microtubule organizational level.
Other advantages, features, and objects of the present invention will become obvious after evaluation of the specification, drawings, and claims.